The major histocompatibility complex (MHC) molecule is a central component of the vertebrate immune system found on the surface of all nucleated cells. The MHC is found in two major forms, namely as MHC class I and class II. Importantly, both versions form functional complexes with proteolytically processed peptides, denoted T cell epitopes, which takes place within the very same cell that expresses the given MHC. The resulting peptide/MHC (pMHC) complex is subsequently found as a transmembrane complex on the surface of the cell—a phenomenon described as antigen presentation. The cell surface-bound pMHC may then interact with its cognate partner—the T cell receptor (TCR), which is found on the surface of T lymphocytes.
Given its pivotal role in adaptive immunity, basic and applied sciences have a substantial interest in understanding the pMHC-TCR interaction at both the cellular and molecular level and thus to have access to recombinant versions of both molecules. It is also an ever growing understanding that the availability of such recombinant molecules is absolutely critical for being able to study and understand the biology of the system, as well as for developing novel therapeutics and diagnostics.
Many significant medical conditions require therapeutic interventions to modulate the activity of the patient's immune system. In e.g. autoimmune diseases and allergies, the overactive immune system and chronic inflammation needs to be suppressed. In contrast, immunostimulation is an approach relevant for infections and cancers to activate and target the immune cells towards the cancerous cells. In addition, transplant recipients usually require immunosuppression. Together, this has lead to the development of a vast amount of immunomodulators, currently a multi Billion dollar industry.
A key to understanding the immunity component in these diseases, and screen for new treatments, is the interaction between the antigen-presenting cells and the T-cells, or more specifically, the interaction between MHC class I and II molecules and the TCR. The class II MHC specifically binds exogenously derived peptides, and presents them to CD4+ T helper cells (TH cells). The TH cell is then activated and becomes an effector cell that secretes various cytokines. These cytokines activate a wide range of other immune cells involved in taking care of the threat. A failure in this system can lead to e.g. an autoimmune disease or to allow cancer cells to survive and divide. Thus, autoimmune diseases are characterized by a strong MHC association and target organ T cell infiltration.
A platform for immunomodulator screening requires stable, fully functional soluble MHC class II molecules. Importantly, the production of soluble MHC class II molecules is currently hampered by a severe problem, namely lack of molecular stability.
In the last few years the ability to produce soluble MHC class I molecules as tetramers (tetramer technology) has revolutionized basic and applied immunology (Constantin et al., 2002, Biological Research for Nursing, 4: 115-127). The reason for this is that tetramer technology has substantially increased the ability to track the course of an immune response in a specific manner both in terms of the antigen and the T cell response, assessed primarily by flow cytometry. This ability has also translated into a much deeper understanding of the immune system and may indeed also give rise to novel diagnostic tools.
To date, tetramer reagents have to a large extent been limited to the MHC class I molecules, as most technical issues regarding recombinant MHC class I production appear to have been solved. Indeed, with regard to MHC class II molecules, this task has proven significantly more challenging. Thus, no general protocol for the production of MHC class II tetramers is at present available, although stand alone examples appear both in the literature and as commercially available reagents (Vollers, S. and Stern, L., 2008, Immunology 123: 305-313). In the few cases where MHC class II tetramers have been available, they have been used extensively, and have had an enormous impact on the understanding of disease development. Thus, given the impact successful recombinant MHC class I production has already shown, it can be seen that there is a strong and clear cut motivation, both academic and commercial, for putting further efforts into novel MHC class II production avenues. However, given the problems encountered to date, success is uncertain.
Due to partially unknown reasons, the MHC class II molecule has proven especially difficult to produce as a stable recombinant molecule in soluble form. The native molecule is a non-covalent transmembrane heterodimer comprising an α- and a β-chain, both of which have transmembrane regions and belong to the immunoglobulin (Ig) superfamily. The extracellular portion of each chain is composed of two domains, each consisting of approximately 90 amino acid residues, of which the two membrane distal domains, the α1 and β1 domains, form an inter-latticed α/β structure essential for the peptide binding property of the T cell epitopes. The two membrane proximal domains, the α2 and the β2 domains, both form discrete Ig domains. In both the α and the β chain, a stretch of approximately 20 amino acid residues spans the cell membrane and on the cytoplasmic side of the membrane a fairly short peptide segment is located.
The dimerization of the α and the β chain is thought to be caused by (i) the transmembrane segments, (ii) peptide binding and (iii) putative accessory components found in the membrane. Hence, once separated from its native context and produced as a soluble molecule, the MHC class II molecule often suffers from intrinsically low stability and very low production levels. Furthermore, extensive and resource demanding case-dependent optimization must be carried out.
In addition, given the above requirements for dimerization, general methods of production of MHC class II molecules in any non-native context, i.e. in any context other than production in association with a membrane of a cell which cell naturally expresses MHC class II molecules, e.g. production as non-soluble molecules displayed on the surface of other biological entities, cells or particles, e.g. as non-soluble molecules expressed on the surface of a phage by way of fusion to viral capsid proteins, is thought to be far from straightforward.
Thus, there is at present no general strategy which exists which allows the production of a stabilized MHC class II heterodimer. However, a variety of studies have been carried out that all represent case-specific successful examples that rather reflect the complexity of the task. These examples are (i) the ectopic expression of a membrane-bound MHC class II heterodimer on the surface of eukaryotic cells by use of a lipid tether (GPI anchor), see Wettstein et al., 1991, J. of Exp. Medicine, 174: 219-228; (ii) the expression of MHC class II ectodomains in insect cells, see Wallny et al., 1995, Eur. J. Immunology, 25: 1262-1266; (iii) the introduction of heterologous dimerization motifs, such as the leucine zipper, C-terminally into the MHC class II molecules, in combination with insect cell production, see Quarsten et al., 2001, J. Immunol., 167: 4861-4868, and Crawford et al., 2006, Immunological Reviews, 210: 156-170; (iv) the production of antibody/MHC class II chimeras in combination with insect cell production, see Casares et al., 1997, Protein Engineering, 10: 1295-1301; (v) the use of bacterial expression systems that allow formation of functional pMHC trimers through refolding approaches from inclusion bodies, see Arimilli et al., 1995, J. Biol. Chem., 270:971-977; or (vi) the use of a truncated bacterial produced single chain MHC class II format which is comprised of the α1 and β1 domains only, that allows formation of functional MHC molecules through refolding approaches from inclusion bodies, see Burrows et al., 1999, Protein Engineering, 12: 771-778. In addition, Landais et al., 2009, J. Immunol., 183:7949-7957, describes an insect cell expression system which uses internal artificial disulphide bridges in conjunction with exogenous leucine zippers for producing stabilized murine I-Ad OVA MHC class II tetramers. Importantly, and despite increased expression levels due to the modification, none of these apparently stabilized-increased molecules exhibited specific T cell staining.